Recent evidence has suggested that qualitative aspects of an anti-tumor T cell response can influence whether tumor rejection can be achieved. However, quantitative aspects of T cell activation also are likely to be critical. Intrinsic to normal T cell responses are multiple negative regulatory pathways that mediate contraction of a clonally expanded T cell pool and subsequently provide inhibitory signals for T cell re-activation. It is reasoned that these mechanisms limit the ability of antigen-expressing tumors to become rejected spontaneously, and likewise preclude effective elimination of established tumors via attempts at immunotherapeutic intervention. A central hypothesis of this proposal is that interfering with or countering these negative regulatory influences on T cell activation may improve that magnitude or duration of an anti-tumor T cell response and thus lead to more efficient tumor elimination. In Specific Aim 1, we will examine the role of the putative negative regulatory receptors Fas, CTLA4, and PD-1 on limiting the efficacy of anti-tumor CD8 + T cell response in vivo. These studies will utilize 2C TCR transgenic/RAG-/- mice interbred with mice deficient in these receptors, alone and in combination. Adoptive transfer of graded numbers of mutant T cells into recipient mice either before tumor challenge or after implantation of a pre-established tumor will enable a quantitative assessment of the contribution of each receptor to be determined. In Specific Aim 2, we will manipulate intracellular signaling pathways to counter death pathways or signaling defects that have been observed in T cell from tumor-bearing mice. Introduction of the anti-apoptotic molecules Bcl-xL and p35, or of CA mutants of the signaling molecules Lck, Ras, or IKKbeta3, will be achieved utilizing adenoviral vectors and CAR transgenic/2C/RAG2 -/- T cells. Adoptive transfer will again be performed and effects on T cell functions and anti-tumor efficacy will be determined. In Specific Aim 3, we will apply new information learned using the TCR transgenic system toward preclinical models of tumor antigen vaccines and adoptive T cell therapy in normal mice. Soluble receptor antagonists, or retroviral transduction of T cells, will be performed to improve immunotherapy of mice in protection assays, treatment of established tumors, and also in mice acquiring spontaneous HPV E7-expressing tumors. The results of these studies will pave the way for future clinical translation of the most effective of these approaches.